Limits...
Whole genome methylation profiles as independent markers of survival in stage IIIC melanoma patients.

Sigalotti L, Covre A, Fratta E, Parisi G, Sonego P, Colizzi F, Coral S, Massarut S, Kirkwood JM, Maio M - J Transl Med (2012)

Bottom Line: A 17 gene methylation signature able to correctly assign prognosis (overall error rate = 0) in stage IIIC patients on the basis of distinct methylation-defined groups was also identified.A discrete whole-genome methylation signature has been identified as molecular marker of prognosis for stage IIIC CM patients.Its use in daily practice is foreseeable, and promises to refine the comprehensive clinical management of stage III CM patients.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cancer Bioimmunotherapy Unit, Centro di Riferimento Oncologico, Istituto di Ricovero e Cura a Carattere Scientifico, Aviano, Italy. lsigalotti@cro.it

ABSTRACT

Background: The clinical course of cutaneous melanoma (CM) can differ significantly for patients with identical stages of disease, defined clinico-pathologically, and no molecular markers differentiate patients with such a diverse prognosis. This study aimed to define the prognostic value of whole genome DNA methylation profiles in stage III CM.

Methods: Genome-wide methylation profiles were evaluated by the Illumina Human Methylation 27 BeadChip assay in short-term neoplastic cell cultures from 45 stage IIIC CM patients. Unsupervised K-means partitioning clustering was exploited to sort patients into 2 groups based on their methylation profiles. Methylation patterns related to the discovered groups were determined using the nearest shrunken centroid classification algorithm. The impact of genome-wide methylation patterns on overall survival (OS) was assessed using Cox regression and Kaplan-Meier analyses.

Results: Unsupervised K-means partitioning by whole genome methylation profiles identified classes with significantly different OS in stage IIIC CM patients. Patients with a "favorable" methylation profile had increased OS (P = 0.001, log-rank = 10.2) by Kaplan-Meier analysis. Median OS of stage IIIC patients with a "favorable" vs. "unfavorable" methylation profile were 31.5 and 10.4 months, respectively. The 5 year OS for stage IIIC patients with a "favorable" methylation profile was 41.2% as compared to 0% for patients with an "unfavorable" methylation profile. Among the variables examined by multivariate Cox regression analysis, classification defined by methylation profile was the only predictor of OS (Hazard Ratio = 2.41, for "unfavorable" methylation profile; 95% Confidence Interval: 1.02-5.70; P = 0.045). A 17 gene methylation signature able to correctly assign prognosis (overall error rate = 0) in stage IIIC patients on the basis of distinct methylation-defined groups was also identified.

Conclusions: A discrete whole-genome methylation signature has been identified as molecular marker of prognosis for stage IIIC CM patients. Its use in daily practice is foreseeable, and promises to refine the comprehensive clinical management of stage III CM patients.

Show MeSH

Related in: MedlinePlus

Validation of microarray data.  Short-term cultures of CM cells generated from neoplastic lesions of 45 stage IIIC melanoma patients were evaluated for WNT10B, TUB, ALOX12B and SLC6A11 methylation and mRNA expression by qMSP and qRT-PCR analyses, respectively. All cells were analyzed at 6th in vitro passage. For each gene, the % of methylation measured by qMSP was defined as the ratio between methylated gene molecules and the sum of methylated and unmethylated gene molecules. Level of gene expression is reported as number of molecules of the target gene normalized to the number of molecules of the housekeeping gene β-actin. Panel a, Correlation between % methylation defined by qMSP and β-values defined by the Illumina HumanMethylation27 Bead-Chip assay was evaluated for each gene through the Spearman’s rank correlation test. Reported P values are two sided. Panel b, Correlation between methylation and mRNA expression was evaluated for each gene by Spearman’s rank correlation test. Reported P values are two sided.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3539917&req=5

Figure 4: Validation of microarray data. Short-term cultures of CM cells generated from neoplastic lesions of 45 stage IIIC melanoma patients were evaluated for WNT10B, TUB, ALOX12B and SLC6A11 methylation and mRNA expression by qMSP and qRT-PCR analyses, respectively. All cells were analyzed at 6th in vitro passage. For each gene, the % of methylation measured by qMSP was defined as the ratio between methylated gene molecules and the sum of methylated and unmethylated gene molecules. Level of gene expression is reported as number of molecules of the target gene normalized to the number of molecules of the housekeeping gene β-actin. Panel a, Correlation between % methylation defined by qMSP and β-values defined by the Illumina HumanMethylation27 Bead-Chip assay was evaluated for each gene through the Spearman’s rank correlation test. Reported P values are two sided. Panel b, Correlation between methylation and mRNA expression was evaluated for each gene by Spearman’s rank correlation test. Reported P values are two sided.

Mentions: Array-based methylation profiling was validated on the short-term CM cell cultures under analysis using quantitative Methylation-Specific PCR (qMSP) for selected genes composing the 17-gene methylation signature. Genes were selected among those having the highest impact on the classification task, which could be identified by their positioning at the top of the shrunken centroids graph (Figure3a). Spearman’s rank correlation identified a highly significant (p ≤ 10-4) positive correlation between methylation values determined by the Illumina Infinium platform and those defined by qMSP, for all of the 4 genes under analysis (WNT10B, TUB, ALOX12B, SLC6A11; Figure4). Coefficients of correlation ranged from 0.541 to 0.791, for WNT10B and ALOX12B, respectively (Figure4).


Whole genome methylation profiles as independent markers of survival in stage IIIC melanoma patients.

Sigalotti L, Covre A, Fratta E, Parisi G, Sonego P, Colizzi F, Coral S, Massarut S, Kirkwood JM, Maio M - J Transl Med (2012)

Validation of microarray data.  Short-term cultures of CM cells generated from neoplastic lesions of 45 stage IIIC melanoma patients were evaluated for WNT10B, TUB, ALOX12B and SLC6A11 methylation and mRNA expression by qMSP and qRT-PCR analyses, respectively. All cells were analyzed at 6th in vitro passage. For each gene, the % of methylation measured by qMSP was defined as the ratio between methylated gene molecules and the sum of methylated and unmethylated gene molecules. Level of gene expression is reported as number of molecules of the target gene normalized to the number of molecules of the housekeeping gene β-actin. Panel a, Correlation between % methylation defined by qMSP and β-values defined by the Illumina HumanMethylation27 Bead-Chip assay was evaluated for each gene through the Spearman’s rank correlation test. Reported P values are two sided. Panel b, Correlation between methylation and mRNA expression was evaluated for each gene by Spearman’s rank correlation test. Reported P values are two sided.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3539917&req=5

Figure 4: Validation of microarray data. Short-term cultures of CM cells generated from neoplastic lesions of 45 stage IIIC melanoma patients were evaluated for WNT10B, TUB, ALOX12B and SLC6A11 methylation and mRNA expression by qMSP and qRT-PCR analyses, respectively. All cells were analyzed at 6th in vitro passage. For each gene, the % of methylation measured by qMSP was defined as the ratio between methylated gene molecules and the sum of methylated and unmethylated gene molecules. Level of gene expression is reported as number of molecules of the target gene normalized to the number of molecules of the housekeeping gene β-actin. Panel a, Correlation between % methylation defined by qMSP and β-values defined by the Illumina HumanMethylation27 Bead-Chip assay was evaluated for each gene through the Spearman’s rank correlation test. Reported P values are two sided. Panel b, Correlation between methylation and mRNA expression was evaluated for each gene by Spearman’s rank correlation test. Reported P values are two sided.
Mentions: Array-based methylation profiling was validated on the short-term CM cell cultures under analysis using quantitative Methylation-Specific PCR (qMSP) for selected genes composing the 17-gene methylation signature. Genes were selected among those having the highest impact on the classification task, which could be identified by their positioning at the top of the shrunken centroids graph (Figure3a). Spearman’s rank correlation identified a highly significant (p ≤ 10-4) positive correlation between methylation values determined by the Illumina Infinium platform and those defined by qMSP, for all of the 4 genes under analysis (WNT10B, TUB, ALOX12B, SLC6A11; Figure4). Coefficients of correlation ranged from 0.541 to 0.791, for WNT10B and ALOX12B, respectively (Figure4).

Bottom Line: A 17 gene methylation signature able to correctly assign prognosis (overall error rate = 0) in stage IIIC patients on the basis of distinct methylation-defined groups was also identified.A discrete whole-genome methylation signature has been identified as molecular marker of prognosis for stage IIIC CM patients.Its use in daily practice is foreseeable, and promises to refine the comprehensive clinical management of stage III CM patients.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cancer Bioimmunotherapy Unit, Centro di Riferimento Oncologico, Istituto di Ricovero e Cura a Carattere Scientifico, Aviano, Italy. lsigalotti@cro.it

ABSTRACT

Background: The clinical course of cutaneous melanoma (CM) can differ significantly for patients with identical stages of disease, defined clinico-pathologically, and no molecular markers differentiate patients with such a diverse prognosis. This study aimed to define the prognostic value of whole genome DNA methylation profiles in stage III CM.

Methods: Genome-wide methylation profiles were evaluated by the Illumina Human Methylation 27 BeadChip assay in short-term neoplastic cell cultures from 45 stage IIIC CM patients. Unsupervised K-means partitioning clustering was exploited to sort patients into 2 groups based on their methylation profiles. Methylation patterns related to the discovered groups were determined using the nearest shrunken centroid classification algorithm. The impact of genome-wide methylation patterns on overall survival (OS) was assessed using Cox regression and Kaplan-Meier analyses.

Results: Unsupervised K-means partitioning by whole genome methylation profiles identified classes with significantly different OS in stage IIIC CM patients. Patients with a "favorable" methylation profile had increased OS (P = 0.001, log-rank = 10.2) by Kaplan-Meier analysis. Median OS of stage IIIC patients with a "favorable" vs. "unfavorable" methylation profile were 31.5 and 10.4 months, respectively. The 5 year OS for stage IIIC patients with a "favorable" methylation profile was 41.2% as compared to 0% for patients with an "unfavorable" methylation profile. Among the variables examined by multivariate Cox regression analysis, classification defined by methylation profile was the only predictor of OS (Hazard Ratio = 2.41, for "unfavorable" methylation profile; 95% Confidence Interval: 1.02-5.70; P = 0.045). A 17 gene methylation signature able to correctly assign prognosis (overall error rate = 0) in stage IIIC patients on the basis of distinct methylation-defined groups was also identified.

Conclusions: A discrete whole-genome methylation signature has been identified as molecular marker of prognosis for stage IIIC CM patients. Its use in daily practice is foreseeable, and promises to refine the comprehensive clinical management of stage III CM patients.

Show MeSH
Related in: MedlinePlus